PROJECT SUMMARY/ABSTRACT Pulmonary lymphatic vessels are thought to be critical for lung function due to the vulnerability of the lungs to edema and their constant exposure to pathogens. Indeed, abnormal pulmonary lymphatics are seen in association with nearly every chronic lung disease including transplant rejection, pulmonary fibrosis, and emphysema, which combined affect nearly 32 million Americans. However, it remains unclear whether these abnormal lymphatics are a consequence of the disease or if lymphatic dysfunction is pathogenic, leaving potential therapeutic targets unexplored. This proposal addresses both an outstanding gap in our ability to study pulmonary lymphatic function and also investigates the basic role of lymphatics in lung disease using two novel mouse models of impaired pulmonary lymphatic flow. The first model uses Clec2-mutant mice, which have severely impaired lymph flow due to an absent platelet plug at the lympho-venous junction and retrograde flow of blood into the lymphatic system that prevents lymphatic drainage. In the second model, lung-specific lymphatic deletion is achieved by inducing diphtheria toxin-mediated cell death of lymphatic endothelial cells in mouse lung transplants. These models show that lymphatic dysfunction leads to accumulation of leukocytes and formation of lymphoid tissue in the lung parenchyma. Significantly, mice with impaired lymphatic flow also have alveolar enlargement and lung injury that resembles human emphysema. In Aim 1 of this proposal, both models will be used to investigate the role of pulmonary lymphatics in leukocyte trafficking and test whether lymphatic function plays a role in immune cell specification and the inflammatory milieu of the lung. Aim 2 will use pulmonary function tests, CT imaging, and expression studies to test the hypothesis that lymphatic dysfunction causes a spontaneous emphysema phenotype in mice, and will investigate the mechanism by which lymphatic dysfunction causes lung injury using rescue studies. These studies are predicted to provide in vivo models for understanding the interplay between lymphatic function, inflammation, and lung injury and may uncover an unappreciated role for lymphatics in emphysema. This proposal also plays a central role in a career development plan for becoming a successful independent investigator focused on vascular biology and disease. The training plan described here provides an opportunity to gain expertise in mouse modeling of human disease, transcriptomics, and physiologic and radiographic analysis of the mouse lung. The University of Pennsylvania is an ideal environment in which to execute this training plan not only because of its excellent physical resources, but also because of its intellectual community of researchers with a track record of strong mentorship of early stage investigators.